The high-resolution three-dimensional structures of G protein coupled receptors (GPCRs) are required in order to understand the mechanisms of ligand binding and activation in this predominant class of transmembrane signaling proteins. In addition, the ability to elucidate the structures of GPCRs would make a significant impact in the design of new drugs, since these receptors are key therapeutic targets for a variety of human diseases. We have made significant progress in the isolation of diffraction-quality crystals for the Beta2 adrenergic receptor (Beta2AR), a Family A GPCR, using a strategy in which a folded, crystallizable domain is integrated into the third intracellular loop. The first specific aim of this proposal is to solve the high-resolution structure of this engineered Beta2AR fusion protein, using all of the strategies for crystal optimization and data collection at our disposal: additives and cryo-protectants will be screened, an advanced microfocus X-ray beamline will be utilized, and if necessary our construct will be further modified. The second specific aim of this proposal is to apply knowledge gained from the fusion protein strategy towards the structural elucidation of the wild-type Beta2AR. This will be accomplished through the formation and crystallization of complexes between the Beta2AR and antibody fragments. A future aim will be to test the strategy of receptor fusion crystallography on other GPCRs. The same domain that was used successfully in the context of the Beta2AR will be cloned into several different therapeutically relevant receptors, which bind to a chemically diverse array of ligands. The expression, pharmacology, and stability of these engineered receptors will be assessed, with the future goal of setting up crystallization trials. Lay summary: Our long-term objective of obtaining crystal structures for GPCRs has broad relevance for public health, in that drugs targeting these receptors are used in the treatment of cardiovascular disease, pulmonary disease, inflammation, diabetes, obesity, and psychiatric disorders. If we can understand the molecular basis for ligand affinity and specificity in GPCR activation, we will gain an important new tool in the design of more effective therapeutics with fewer side-effects. [unreadable] [unreadable] [unreadable]